The present invention relates generally to G-protein, seven-transmembrane-receptor-like polynucleotides and polypeptides, as well as modulators of the interaction between the polypeptides and their ligand(s) that are useful for inhibiting the neurological activity of organisms, e.g., invertebrates.
It has been estimated that as many as one in three humans is infected by one or more species of parasitic helminth (flatworms and roundworms). Helminths also represent a chronic and continuing threat to the health of livestock and companion animals; their abundance and resistance to anthelmintic drugs effectively prevents animal agriculture in certain environments. Despite the prevalence of helminth-caused disease, little is known of helminth physiology. This ignorance, in turn, hampers efforts to identify agents capable of controlling these pathogens.
Flatworms (platyhelminths) are evolutionarily quite distinct from the roundworms (nematodes) and differ markedly in neuromuscular anatomy and physiology. Only a subset of one class of drugs (some of the benzimidazoles) shows activity against both flatworms and roundworms. The diversity among the animal species that threaten the health of man, livestock, crops, and sensitive environmental niches, presents a challenge to efforts to mount broad-based attacks on the pest organisms.
G protein-coupled receptors (i.e., GPCRs) form a vast superfamily of cell surface receptors which are present in virtually all animal cells and are characterized by an amino-terminal extracellular domain, a carboxy-terminal intracellular domain, and a serpentine structure that passes through the cell membrane seven times. Hence, such receptors are sometimes also referred to as seven transmembrane (7TM) receptors. These seven transmembrane domains define three extracellular loops and three intracellular loops, in addition to the amino- and carboxy-terminal domains. The extracellular portions of the receptor have a role in recognizing and binding one or more extracellular binding partners (e.g., ligands), whereas the intracellular portions have a role in recognizing and communicating with downstream effector molecules.
The G protein-coupled receptors bind a variety of ligands including calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and even photons. Not surprisingly, the GPCRs are important in the normal (and sometimes the aberrant) function of many cell types. [See generally Strosberg, Eur. J. Biochem., 196: 1-10 (1991) and Bohm et al., Biochem J., 322: 1-18 (1997).] When a specific ligand binds to its corresponding receptor, the ligand typically stimulates the receptor to activate a specific heterotrimeric guanine nucleotide-binding regulatory protein (G protein) that is coupled to the intracellular portion or region of the receptor. The G protein, in turn, transmits a signal to an effector molecule within the cell by either stimulating or inhibiting the activity of that effector molecule. These effector molecules include adenylate cyclase, phospholipases and ion channels. Adenylate cyclase and phospholipases are enzymes that are involved in the production of the second messenger molecules cAMP, inositol triphosphate and diacyglycerol. It is through this sequence of events that an extracellular ligand stimulus exerts intracellular changes through a G protein-coupled receptor. Each such receptor has its own characteristic primary structure, expression pattern, ligand binding profile, and intracellular effector system.
Because of the vital role of G protein-coupled receptors in the communication between cells and their environment, such receptors are attractive targets for therapeutic intervention, and drugs that activate or antagonize the activation of such receptors are known. For receptors having a known ligand, the identification of agonists or antagonists may be sought specifically for mimicking, enhancing or inhibiting the action of the ligand. Thus, GPCRs show promise as potential targets of methods for treating infestations and/or infections caused by a variety of invertebrate pests, including both ecto- and endo-parasites. However, such methods must be able to discriminate the GPCRs of the invertebrate pest organisms from the GPCRs found in those species of plants and vertebrate animals upon whom the pests prey.
A large family of peptides (typically 4-15 amino acids in length) that is largely, if not exclusively, found in invertebrate animals such as helminths is a class of neuropeptides known as FMRFamide related peptides (i.e., FaRPs). The prototypical FMRFamide peptides are so named because of the xe2x80x9cFMRFxe2x80x9d amino acid sequence, including the consensus xe2x80x9cRFxe2x80x9d sequence, at their C-termini. As neuropeptides, these molecules are involved in vital biological processes requiring controlled neuromuscular activity. Although some neurotransmitters and neuromodulators (including neuropeptides) have been shown to function as ligands for receptors, to date there has been no identification of a FaRP neuropeptide as a ligand of a GPCR.
Because of the toxic potential of broad-spectrum chemical parasiticides, there exists a need in the art for targeted biologicals capable of selectively interfering with the life cycle of harmful invertebrates such as helminths and insects without harming host plant and animal species, as well as the environment.
The present invention generally relates to materials and methods for the targeted interference with vital biological processes of pest invertebrates. By providing materials and methods for modulating the activity of invertebrate GPCRs involved in neuromuscular activity, the invention provides a biological approach to invertebrate pest control that can minimize the deleterious consequences to non-pest species of animals, including man, as well as plants and the environment in general.
One aspect of the invention is a screening method for identifying candidate anti-invertebrate modulators that affect one or more activities of an invertebrate GPCR-like receptor involved in neuromuscular functioning including, e.g., binding of a GPCR-like receptor to a ligand, typically a peptide ligand, and signal transduction. The method comprises the steps of: (a) contacting a test compound with a composition, wherein the composition contains a GPCR-like receptor encoded by a polynucleotide having a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 104, 106, 108, 110, 112, 114, 116, 176 and 178, or a polynucleotide hybridizing to the GPCR-like receptor under stringent conditions of hybridizing at 42xc2x0 C. in a solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate, and washing twice for 30 minutes at 60xc2x0 C. in a wash solution comprising 0.1xc3x97SSC and 1% SDS; and (b) measuring the activity of the GPCR-like receptor in the presence and absence of the test compound. In preferred embodiments, the screening method includes a receptor, or fragment, variant or derivative thereof, having a sequence set forth in SEQ ID NOS: 7, 21, 25, 35, 43, 45, 105, 107, 109, 111, 113, 115, 117, 177 and 179.
As one of ordinary skill in the art would recognize, the above-described method can be practiced with a variety of GPCR-like receptors. For example, the GPCR-like receptor used in the screening method may be encoded by a polynucleotide having a sequence set forth in any one of SEQ ID NOS:43, 21, 45, 35, 7, 106, and 104. As noted above, such GPCR-like receptors may be used in screening assays designed to measure a GPCR-like receptor activity, including binding activity. Expressly contemplated are embodiments of the screening method comprising a GPCR-like receptor encoded by a polynucleotide comprising a sequence set forth in SEQ ID NO:43 and a peptide comprising a sequence selected from the group consisting of SEQ ID NOS:85, 86, 88, 89, and 118, wherein the peptide binds to the GPCR-like receptor. encoded by a polynucleotide having the sequence set forth in SEQ ID NO:43 is provided. In an alternative embodiment, the GPCR-like receptor comprises a sequence set forth in SEQ ID NO:21 and the peptide comprises a sequence selected from the group consisting of SEQ ID NOS:78, 79, 80, 84, 87, 92, 98, 100, 120, 171, 143, 122, 123, 97, 85, 83, 101, 102, 93, 88, 91, 94, 93, 90, 152, 153, 154, 155, 156, 157, 80, 158, 119, 159, 160, 161, 162, 163 and 164. Another embodiment involves a GPCR-like receptor comprising a sequence set forth in SEQ ID NO:45 and a peptide comprising a sequence selected from the group consisting of SEQ ID NOS:86, 118, 125, 88, 126, 127, 128, 129, 102, 131, 100, 133, 92, 135, 136, 137, 87, 139, 91, 141 and 83. In yet another embodiment, the GPCR-like receptor comprises a sequence set forth in SEQ ID NO:35 and a peptide comprising a sequence selected from the group consisting of SEQ ID NOS:99, 97, 96, 77, 82, 81, 87, 100, 92, 80, 98, 120, 121, 79 and 84. In still another embodiment, the GPCR-like receptor comprises a sequence set forth in SEQ ID NO:7 and a peptide comprising a sequence selected from the group consisting of SEQ ID NOS:94, 103, 95, 101, 85, 79, 84, 87, 86, 80, 92, 100, and 180. Yet another embodiment involves a GPCR-like receptor comprising a sequence selected from the group consisting of SEQ ID NO:106 and a peptide comprising a sequence selected from the group consisting of SEQ ID NOS:80, 92, 98, 100, 120, 121, 79, 84, 136, 87 and 86. Still another one of the many embodiments of this aspect of the invention involves a GPCR-like receptor comprising a sequence set forth in SEQ ID NO:104 and a peptide comprising a sequence selected from the group consisting of SEQ ID NOS:80, 92, 98, 100, 120, 121, 79, 84, 136, 87, 86, 150, 151, 133, 165, 91, 166, 131 and 167.
Another aspect of the invention is a method of identifying an anti-invertebrate modulator of an activity of an invertebrate GPCR-like receptor comprising the following steps: (a) contacting a test compound and a composition, wherein the composition contains a GPCR-like receptor selected from the group consisting of polypeptides encoded by a polynucleotide having a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 104, 106, 108, 110, 112, 114, 116, 176 and 178, or a polynucleotide hybridizing to the GPCR-like receptor under stringent conditions of hybridizing at 42xc2x0 C. in a solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes at 60xc2x0 C. in a wash solution comprising 0.1xc3x97SSC and 1% SDS, optionally in the presence of a peptide or other ligand of the receptor; and (b) measuring the activity of the GPCR-like receptor in the presence and absence of the test compound. Modulators are identified as test compounds that alter (i.e., increase or decrease) a GPCR-like receptor function, such as a binding property of a receptor or an activity such as G protein-mediated signal transduction or membrane localization. The composition may contain an isolated GPCR-like receptor; alternatively, the composition may contain a GPCR-like receptor in association with, e.g., an intact cell or cell portion, such as a membrane. Presently preferred embodiments of the method use a GPCR-like receptor having an amino acid sequence set forth in SEQ ID NOS: 8, 22, 26, 36, 44, 105, 107, 109, 111, 113, 115, 117, 177 or 179. Preferred peptides are neuropeptides derived from invertebrates and include the FaRP family of neuropeptides. Particularly preferred are invertebrate neuropeptides having an amino acid sequence selected from the group consisting of SEQ ID NOS: 77-103 and 118-151. The methods of the invention embrace neuropeptides that are attached to a label, such as a radiolabel (e.g., 125I, 35S, 32P, 33P, 3H), a fluorescence label, a chemiluminescence label, an enzymic label and an immunogenic label.
In various embodiments of the method, the assay may take the form of an ion flux assay, a yeast growth assay, a non-hydrolyzable GTP assay such as a [35S]-GTPxcex3S assay, a cAMP assay, an inositol triphosphate assay, a diacylglycerol assay, an Aequorin assay, a Luciferase assay, a FLIPR assay for intracellular Ca2+ concentration, a mitogenesis assay, a MAP Kinase activity assay, an arachidonic acid release assay (e.g., using [3H]-arachidonic acid), and an assay for extracellular acidification rates, as well as other binding or function-based assays of GPCR activity that are generally known in the art. In several of these embodiments, the invention comprehends the inclusion of any of the G proteins known in the art, such as Gxcex116, Gxcex115, or chimeric Gqi5, Gqs5, Gqo5, or Gqz5.
In another aspect of the invention, a method of identifying a candidate anti-invertebrate modulator is provided. The method comprises the steps of: (a) contacting a test compound and a composition, wherein the composition contains a GPCR-like receptor encoded by a polynucleotide selected from the group consisting of receptor polyucleotides having a sequence set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 104, 106, 108, 110, 112, 114, 116, 176 and 178, and polynucleotides hybridizing to the receptor polynucleotides under stringent conditions of hybridizing at 42xc2x0 C. in a solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate, and washing twice for 30 minutes at 60xc2x0 C. in a wash solution comprising 0.1xc3x97SSC and 1% SDS; and (b) identifying a test compound that binds to or interacts with the composition as a candidate anti-invertebrate modulator. In a preferred embodiment, the polynucleotide encoding the GPCR-like receptor comprises a sequence selected from the group consisting of SEQ ID NOS: 7, 21, 25, 35, 43, 45, 105, 107, 109, 111, 113, 115, 117, 177 and 179.
Another aspect of the invention is a method of identifying an anti-invertebrate agent comprising the following steps: (a) identifying a modulator using the method described above; (b) contacting the modulator and an invertebrate tissue; and (c) measuring the response of the invertebrate tissue, e.g. neural signaling or neuromuscular activity, thereby identifying the modulator as an anti-invertebrate agent. Although any invertebrate tissue may be used in the method, presently preferred tissue sources are helminths and insects. An exemplary type of suitable invertebrate tissue is neuromuscular tissue, e.g. in isolated form or remaining in association with part, or all, of an invertebrate organism. The anti-invertebrate agents, and compositions comprising one of those agents, identified by this method constitute yet another aspect of the invention.
In a related aspect, the invention provides a method for treating an invertebrate comprising the step of contacting the invertebrate with a biologically effective amount of a modulator identified by the methods described herein. A biologically effective amount of a modulator is an amount that is sufficient to induce a desired response in the treated invertebrate. Thus, a biologically effective amount of a modulator may be the amount that interferes with physiological activity of the treated invertebrate in a non-lethal manner (i.e., a biostatic effect) or in a lethal manner (i.e., a biocidal effect). A preferred modulator for use in the treatment methods is an inhibitor of GPCR-like receptor activity. The invention is not limited to particular means for delivering the modulator to an invertebrate, nor is the invention limited as to the compositions comprising the modulator which may be delivered.
Another aspect of the invention is drawn to methods of producing an invertebrate GPCR-like receptor comprising the following steps:(a) incubating a source cell at a temperature of at least about 35xc2x0 C.; (b) lowering the temperature to at most about 26xc2x0 C.; and (c) detecting the GPCR-like receptor. In various embodiments, the temperatures may be varied to optimize production using no more than routine experimentation. For example, the cells may be incubated at temperatures higher than about 35xc2x0 C., e.g., a temperature of at least about 37xc2x0 C.; the temperature also may be lowered beyond 30xc2x0 C., for example to at most 29xc2x0 C., at most 28xc2x0 C., at most 27xc2x0 C., at most 26xc2x0 C., at most 25xc2x0 C. or at most 24xc2x0 C. In some embodiments, both the incubation temperature may be raised above 35xc2x0 C. and the temperature lowering may extend beyond 30xc2x0 C. The method of producing an invertebrate GPCR-like receptor may further comprise recovering the GPCR-like receptor, which may be native or recombinant in origin. The receptor may be recovered in intact cells, cell portions (e.g., membranes) obtained as a result of cell lysis, or in isolated form. Any of a wide variety of cells may be used as source cells, such as cells derived from mammals, amphibians, arthropods (e.g., insects), mollusks, helminths, and others. It is anticipated that this method of producing a GPCR-like receptor is particularly suited to the recombinant production of GPCR-like receptors using non-invertebrate cells, such as mammalian cells.
The invention also comprehends compositions of matter, such as a modulator of an activity of the GPCR-like receptors identified by the methods described herein. The modulators of the invention exhibit a variety of chemical structures, which can be generally grouped into non-peptide mimetics of natural GPCR-like receptor ligands, peptide and non-peptide allosteric effectors of GPCR-like receptors, and peptides and non-peptide compounds that function as activators or inhibitors (competitive, uncompetitive and non-competitive) (e.g., antibody products) of an ultimate GPCR-like receptor activity. For example, such modulators may be compounds or compositions that are agonists or antagonists of ligand binding to a GPCR-like receptor, allosteric effectors thereof, or compounds (or compositions) that affect the ultimate activity of GPCR-like receptors through direct action on the receptor or an effect introduced downstream of the receptor in, e.g., a signal cascade. In addition, modulators according to the invention may be compounds or compositions that interfere with the expression of a GPCR-like receptor, either through inhibiting transcription of the DNA or translation of the corresponding mRNA. Expression of the GPCR-like receptor can be monitored by any methods known in the art, including Western blot analysis using polyclonal or monoclonal antibodies to the GPCR-like receptor or Northern blot analysis or quantitative polymerase chain reaction (PCR) using suitable probes or primers based on the sequence of the GPCR-like receptor gene. In particular, modulators that interfere with the expression of gene products include anti-sense polynucleotides and ribozymes that are complementary to the gene sequences. The invention further embraces modulators that affect the transcription of gene products of the invention through the formation of oligonucleotide-directed triplet helix formation. The invention does not restrict the sources for suitable modulators, which may be obtained from natural sources such as plant, animal or mineral extracts, or non-natural sources such as small molecule libraries, including the products of combinatorial chemical approaches to library construction, and peptide libraries. Preferred peptide receptors have an amino acid sequence selected from the group consisting of SEQ ID NOS: 8, 22, 26, 36, 44, 105, 107, 109, 111, 113, 115, 117, 177 or 179.
Another aspect of the invention is drawn to an isolated GPCR-like receptor comprising a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 105, 107, 109, 111, 113, 115, 117, 177, and 179, and variants and fragments thereof. Preferred receptors have sequences set forth in SEQ ID NOS: 8, 22, 26, 36, 44, 105, 107, 109, 111, 113, 115, 117, 177 and 179. Variants and fragments of GPCR-like receptors retain at least one biological or immunological property of the cognate GPCR-like receptor, and are at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 225, 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, or at least 450 amino acids in length. Fragments specifically include GPCR domains of the receptors. Biological activities of GPCR-like receptors according to the invention include, but are not limited to, the binding of a natural or an unnatural ligand, as well as any one of the functional activities of GPCRs known in the art. Other non-limiting examples of GPCR activities include transmembrane signaling of various forms, which may involve G protein association and/or the exertion of an influence over G protein binding of various guanidylate nucleotides; transmembrane localization; or binding of accessory proteins or polypeptides unrelated to known G proteins.
The invention also provides an isolated polynucleotide encoding a GPCR-like receptor. Such polynucleotides may be selected from the group consisting of: (a) a polynucleotide comprising a nucleotide sequence encoding any one of the amino acid sequences set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 105, 107, 109, 111, 113, 115 117, 177, and 179 (including the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 104, 106, 108, 110, 112, 114, 116, 176, and 178); and (b) a polynucleotide which hybridizes under conditions of high stringency to the complement of the polynucleotide of (a). Exemplary conditions of high stringency are provided below. Such polynucleotides also include polynucleotides that exhibit at least 90%, at least 95%, at least 98%, at least 99% or at least 99.9% sequence identity to either a polynucleotide sequence disclosed in the sequence listing (i.e., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 104, 106, 108, 110, 112, 114, 116, 176 and 178) or to a polynucleotide encoding a GPCR-like receptor comprising one of the sequences disclosed in the sequence listing (i.e., SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 105, 107, 109, 111, 113, 115, 117, 177, and 179). Any one of the publicly available algorithms (e.g., the BLAST1 program of GCG) for comparing sequences may be used in determining the degree of sequence similarity (including appropriate penalties for gap introductions). A preferred algorithm is the BLAST algorithm implemented at the GenBank website under the auspices of the National Center for Biotechnology Information A on the World Wide Web at ncbi.nlm.nih.gov/BLAST/ using default parameters. A polynucleotide of the invention may be partially or wholly chemically synthesized and embraces an anti-sense polynucleotide which specifically hybridizes to the complement of one or more of the above-described polynucleotides. In related aspects, the invention comprehends vectors comprising these polynucleotides preferably operably linked to expression control sequences, including expression vectors, as well as non-native host cells transformed or transfected with a polynucleotide in accordance with the invention or a host cell transformed or transfected with a vector of the invention. All suitable native and non-native host cells are embraced by the invention, including mammalian cells (e.g., COS cells, CHO cells, HEK293 cells), insect cells (e.g., Drosophila melanogaster S2 cells, Spodoptera frugiperda Sf9 cells, High-5 cells), yeast cells, bacterial cells (e.g., E. coli) and helminthic cells. The suitability of a particular cell for use as a host cell in accordance with the invention will depend on the ability to introduce a polynucleotide of the invention into the cell by any known means of transformation or transfection. Preferred host cells will also be capable of stably maintaining the introduced polynucleotide and will present a minimum of obstacles to propagation.
Another aspect of the invention is directed to a genetically modified invertebrate comprising a polynucleotide encoding a heterologous GPCR-like receptor (e.g., transgene) as described above or comprising a modification in a native gene encoding a GPCR-like receptor (e.g., an insertional disruption or deletion of the gene). The GPCR-like receptor encoding gene may be expressed at normal levels for that gene or may be overexpressed or underexpressed. A preferred invertebrate for generation of genetically modified organisms is a member of the helminths.
Yet another aspect of the invention is drawn to diagnostic methods for determining neurological abnormalities associated with aberrant GPCR-like receptor activity. Such methods specifically measure the presence, and optionally quantity, of a GPCR-like receptor polynucleotide according to the invention or the presence, and optionally quantity or activity, of a GPCR-like receptor. Any method known in the art may be used to measure the specific polynucleotides or polypeptides of the invention, and measurements may be performed on intact organisms, isolated tissues, or cell cultures. In a related aspect, the invention contemplates methods for diagnosing invertebrate infestation of an organism (e.g., mammals such as humans, mammalian livestock, other vertebrates such as fish, and non-pest invertebrates such as molluscs) or an environment using a specific measurement of a polynucleotide or polypeptide according to the invention.
Another aspect of the invention is directed to treatment methods. The invention contemplates methods of killing or inhibiting the viability of invertebrates using the modulators described above or identified as described above, including antibodies and antisense polynucleotides. Such methods include methods for treating infestations and/or infections caused by a variety of invertebrate pests, including both ecto- and endo-parasites. A variety of human and other animal ailments, particularly those ailments relating to aberrant neurological functioning, are treated by administering a biologically effective amount of a modulator, GPCR-like receptor polynucleotide or GPCR-like receptor to a cell, tissue, organism, or environment using techniques known in the art.
Use of such modulators in the preparation of a medicament for treating parasitic infection is also contemplated. A variety of administration regimens known in the art are available to deliver a therapeutically effective (i.e., a level of activity capable of deleteriously affecting at least one biological process of a parasite, or more generally, of a pest organism) activity level of a modulator, directly or indirectly, to a parasite or pest, or to an environment associated with the parasite or pest.
Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the preferred embodiments of the invention.
The present invention has several aspects, one of which is modulating the activity of invertebrate G protein-coupled receptors, which is useful in controlling a wide variety of pest organisms that affect the health of humans and other animals such as domestic pets and livestock, as well as plants. The G protein-coupled receptors typically transduce signals involved in the neurophysiological functioning of the target invertebrates and interference with that functioning frequently proves fatal to the target organism. Moreover, these receptors are found only in invertebrates, so that specific modulators are safe for use around humans, as well as their pets, livestock, and crops.
A xe2x80x9cGPCR-like receptorxe2x80x9d is a polypeptide receptor exhibiting the structural characteristics of G protein-coupled receptors (GPCRs), i.e., an N-terminal extracellular region, comprising several loop-like domains (typically three), a transmembrane region comprising seven transmembrane domains arranged in a typical serpentine disposition, and an intracellular region comprising several loop-like domains (typically three).
Invertebrate neuropeptides show profound neuromuscular effects and FaRPs represent the largest family of such neuropeptides known to date. A xe2x80x9cFaRPxe2x80x9d is a xe2x80x9cFMRFamide-related peptide.xe2x80x9d A xe2x80x9cFMRFamide,xe2x80x9d in turn, is a relatively small peptide, typically having 4-15 amino acids, that matches at least three of the four listed amino acids (FMRF) at its C-terminus. Generally, FaRPs exhibit neurophysiological effects and are therefore properly grouped in a class of neuropeptides that frequently function as ligands of GPCR-like receptors. A xe2x80x9cmodulatorxe2x80x9d is an effector of a GPCR-like receptor function, which include the binding of one or more ligands, localization to a membrane, and signal transduction. Signal transduction may involve a change in the relative affinities of a GPCR-associated G protein for various guanidylate nucleotides. With respect to polynucleotides and polypeptides of the invention, xe2x80x9csynthesized,xe2x80x9d as used herein and understood in the art, refers to polynucleotides or polypeptides produced by purely chemical, as opposed to enzymatic, methods. xe2x80x9cWhollyxe2x80x9d synthesized sequences are therefore produced entirely by chemical means, and xe2x80x9cpartiallyxe2x80x9d synthesized sequences embrace those wherein only portions of the resulting polynucleotide or polypeptide were produced by chemical means.
The present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, including splice variants thereof) encoding invertebrate G protein-coupled receptors. DNA polynucleotides of the invention include genomic DNA, cDNA, and DNA that has been chemically synthesized in whole or in part.
Genomic DNA of the invention comprises the protein coding region for a polypeptide of the invention and includes allelic variants thereof. It is widely understood that, for many genes, genomic DNA is transcribed into RNA transcripts that undergo one or more splicing events wherein intron (i.e., non-coding regions) of the transcripts are removed, or xe2x80x9cspliced out.xe2x80x9d RNA transcripts that can be spliced by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode the same polypeptide, are referred to in the art as splice variants. Splice variants therefore are encoded by the same original genomic DNA sequences but arise from distinct mRNA transcripts found in at least one cell. By way of non-limiting example, several embodiments of the invention are characterized by one of the particular splice variants identified in Table 1, below.
Allelic variants are modified forms of a wild-type gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants which arise from in vitro manipulation), and are also comprehended by the invention.
The invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encoding invertebrate GPCR-like receptors (conventionally followed by second strand synthesis of a complementary strand to provide a double-stranded DNA). In addition to cDNA forms of the polynucleotides identified in Table 1 as splice variants, preferred cDNAs according to the invention include those polynucleotides having sequences selected from the group consisting of SEQ ID NOS: 13, 15 and 21. These sequences differ from sequences in the Wormpep database that exhibit some similarity thereto, as shown in Table 2.
Preferred DNA sequences encoding invertebrate GPCR-like receptor polypeptides are set out in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 104, 106, 108, 110, 112, 114, 116, 176 and 178. A preferred DNA of the invention comprises a double stranded molecule (for example, the molecule having one of the sequences set forth in the above-referenced SEQ ID NOs, along with the complementary molecule (the xe2x80x9cnon-coding strandxe2x80x9d or xe2x80x9ccomplementxe2x80x9d) having a sequence unambiguously deducible from one of those sequences, according to Watson-Crick base-pairing rules for DNA). Also preferred are other polynucleotides encoding one of the invertebrate GPCR-like receptor polypeptides of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 105, 107, 109, 111, 113, 115, 117, 177, and 179 which may differ in sequence from the corresponding polynucleotides of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 104, 106, 108, 110, 112, 114, 116, 176 and 178 by virtue of the well-known degeneracy of the universal genetic code.
The invention further embraces invertebrate species (preferably helminth and insect) homologs of the disclosed GPCR-like DNAs. Species homologs, sometimes referred to as xe2x80x9corthologs,xe2x80x9d in general share at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% similarity with the DNA sequences disclosed herein. Percent sequence xe2x80x9csimilarityxe2x80x9d with respect to polynucleotides of the invention is defined herein as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the relevant sequences set forth in the sequence listing below, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Preferred for comparative sequence analyses is the BLAST program available from GCG, implemented with default parameters. Also preferred is the Blastall program available from NCBI.
The polynucleotide sequence information provided by the invention makes possible large scale expression of an encoded polypeptide by techniques well known and routinely practiced in the art. Polynucleotides of the invention also permit identification and isolation of polynucleotides encoding related GPCR-like polypeptides, such as the aforementioned allelic variants and species homologs, by well known techniques including Southern and/or Northern hybridization, and by polymerase chain reaction (PCR). Examples of related polynucleotides include polynucleotides encoding polypeptides homologous to the invertebrate GPCRs and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of those GPCRs. Invertebrate genes encoding proteins homologous to the disclosed GPCRs can also be identified by Southern and/or PCR analysis and are useful in methods of the invention described below. Knowledge of the sequence of the disclosed GPCR-encoding DNAs also makes possible, through use of Southern hybridization, polymerase chain reaction (PCR), and other known techniques, the identification of genomic DNA sequences encoding GPCR expression control regulatory sequences such as promoters, operators, enhancers, repressors, and other regulatory sequences known in the art. Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express an invertebrate GPCR.
The disclosure herein of a full-length polynucleotide encoding invertebrate GPCR-like polypeptides makes readily available to the worker of ordinary skill in the art every possible fragment of the full-length polynucleotides. The invention therefore provides fragments of invertebrate GPCR-encoding polynucleotides comprising at least 14-15, and preferably at least 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotide encoding an invertebrate GPCR.
Fragment polynucleotides contemplated by the invention encode immunologically active peptides capable of interacting with specific anti-GPCR antibodies, as well as domains of GPCRs. The GPCR domains (i.e., the N-terminal extracellular domain, one or more of the three extracellular loop domains, one or more of the seven transmembrane domains, one or more of the three intracellular loop domains, or the C-terminal intracellular domain) of invertebrate GPCRs disclosed herein are characterized in Example 2 and Table 6, below. Full length or fragment polynulcleotides can be linked to polynucleotides encoding heterologous polypeptides, e.g., for producing variants that are fusion proteins.
Preferably, fragment polynucleotides of the invention comprise sequences unique to one of the disclosed GPCR-encoding sequences, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., xe2x80x9cspecificallyxe2x80x9d) to polynucleotides encoding a disclosed invertebrate GPCR (or fragments thereof) and not to polynucleotides encoding other GPCRs. Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also fragments of the full-length sequence derived from introns, regulatory regions, and/or other non-translated sequences. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases. Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize. Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent, and enzymatic labeling.
Fragment polynucleotides are particularly useful as probes for detection of full-length invertebrate GPCR-like polynucleotides, or other polynucleotide fragments thereof. One or more fragment polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding an invertebrate GPCR-like receptor polypeptide, or used to detect variations in a polynucleotide sequence encoding such a polypeptide.
The invention also embraces DNAs encoding invertebrate GPCR-like polypeptides that hybridize under conditions of moderate or high stringency to the non-coding strand, or complement, of a polynucleotide comprising one of the sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 104, 106, 108, 110, 112, 114, 116, 176 and 178.
Exemplary conditions of high stringency are as follows: hybridization at 42xc2x0 C. in a solution (i.e., a hybridization solution) comprising 50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes at 60xc2x0 C. in a wash solution comprising 0.1xc3x97SSC and 1% SDS. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration, as described in Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley and Sons (1994), pp.6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51.
The invention also provides a purified and isolated invertebrate GPCR-like polypeptide encoded by a polynucleotide of the invention. Presently preferred is a polypeptide comprising one of the amino acid sequences set out in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 105, 107, 109, 111, 113, 115, 117, 177, and 179, and species homologs thereof. The invention also embraces a GPCR-like polypeptide encoded by a DNA selected from the group consisting of: (a) the DNA sequence set out in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 104, 106, 108, 110, 112, 114, 116, 176 and 178 and species homologs thereof, and (b) a DNA molecule encoding a GPCR-like gene product that hybridizes under conditions of moderate or high stringency to the DNA of (a). The invention further embraces polypeptides that have at least about 99%, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, and at least about 50% identity and/or homology to the preferred polypeptides of the invention. Percent amino acid sequence xe2x80x9cidentityxe2x80x9d with respect to the preferred polypeptides of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the GPCR-like gene product sequence after aligning both sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent sequence xe2x80x9chomologyxe2x80x9d with respect to the preferred polypeptides of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in one of the GPCR-like receptor polypeptide sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and also considering any conservative substitutions as part of the sequence identity. Conservative substitutions are defined as set out in Tables 3 and 4.
GPCR polypeptides of the invention may be isolated from natural invertebrate cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. GPCR-like gene products of the invention may be full-length polypeptides, biologically active fragments, or variants thereof which retain specific biological or immunological activity. Variants may comprise GPCR-like polypeptide analogs wherein one or more of the specified (i.e., naturally encoded) amino acids is deleted or replaced or wherein one or more non-specified amino acids are added: (1) without loss of one or more of the biological activities or immunological characteristics specific for the GPCR-like receptor; or (2) with specific disablement of a particular biological activity of the GPCR-like polypeptide. Contemplated deletion variants also include fragments lacking portions of a GPCR-like polypeptide not essential for biological activity, and insertion variants include fusion polypeptides in which the wild-type GPCR-like polypeptide or fragment thereof has been fused to another polypeptide.
Variant GPCR-like polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention. Conservative substitutions are recognized in the art to classify amino acids according to their related physical properties and can be defined as set out in Table 3. (See, WO 97/09433, page 10, published Mar. 13, 1997 (PCT/GB96/02197, filed Sep. 6, 1996).) Alternatively, conservative amino acids can be grouped as defined in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-77] as set out in Table 4.
Variant GPCR-like polypeptides of the invention include mature GPCR-like gene products, i.e., wherein leader or signal sequences are removed, having additional amino terminal residues. GPCR-like gene products having an additional methionine residue at position-1 are contemplated, as are GPCR-like receptors having additional methionine and lysine residues at positions-2 and -1. Variants of these types are particularly useful for recombinant protein production in bacterial cell types. Variants of the invention also include gene products wherein amino terminal sequences derived from other proteins have been introduced, as well as variants comprising amino terminal sequences that are not found in naturally occurring proteins.
Another component useful in some methods for identifying modulators of GPCR-like receptor activity, described below, is a class of neuropeptides that includes FaRPs. The FaRPs comprise a large family of peptides that typically function as neurotransmitters in the invertebrate nervous system. The FaRPs that are associated with these neurophysiological functions generally are about four to about nine amino acid residues in length and include a C-terminal amino acid sequence motif of (aromatic)-X-R-Famide, where X is typically M, L, I or V. Day et al., Peptides 20:999-1019 (1999), which is incorporated by reference in its entirety. However, peptides that are exceptions to this rule are also contemplated by the invention, particularly those that are co-encoded on FaRP precursor genes or those that share the characteristics of conforming peptides. Also contemplated by the invention are neuropeptide variants, including FaRP variants, peptides exhibiting an RYamide motif, and other peptides such as retro-inverso neuropeptides (e.g., FaRPs comprising the D stereoisomers of amino acids in a sequence that is reversed from a reference FaRP). For example a retro-inverso variant of AL-FL-ML-RL-FL would be FD-RD-MD-FD-AD.
Neuropeptides such as FaRPs have been found in a wide variety of invertebrates, including arthropods such as insects (e.g., locusts and flies such as Drosophila) and lobsters, mollusks such as the snail Lymnaea stagnalis, and helminths such as nematodes (e.g., C. elegans, A. suum, Haemonchus contortus), trematodes (e.g., Schistosoma mansoni) and cestodes, among others, including Manduca. Information relating to the structure, function, and structure-function relationships ofthese neuropeptides is known in the art [Day et al. (1999)]. Table 5 below provides a classification of several known C. elegans FaRPs, which are examples of the class of neuropeptides useful in practicing the invention.
Further characterization of the structure-function relationships of neuropeptides, such as FaRPs, that are embraced by the invention may be readily accomplished by one of ordinary skill in the art. For example, amino acid-scan modifications (e.g., alanine-scan), in which each residue is sequentially replaced with another amino acid such as alanine are available. Additionally, given the knowledge in the art (see generally, Geary et al., 1999), such modifications to known neuropeptides (e.g., FaRPs) as the internal or terminal deletion of one or more amino acids, as well as the internal or terminal addition of residues, involves no more than routine experimentation. Such xe2x80x9cvariantsxe2x80x9d of FaRPs and related neuropeptides are among the neuropeptides contemplated for use in the methods of the invention.
The GPCR-like receptor polypeptides and neuropeptides produced by the methods described above are useful in assays for modulators of GPCR-like receptor activities, including binding partner (e.g., ligand) binding. Assays contemplated by the invention include both binding assays and activity assays; these assays may be performed in conventional or high throughput formats. GPCR-like receptor activity is defined as including the binding of any binding partner, such as a ligand, as well as the propagation of any transmembrane signal (e.g., stimulation of a G protein or influence on the flux of an ion across a membrane). Modulator screens are designed to identify stimulatory and/or inhibitory agents. The sources for potential agents to be screened include natural sources, such as a cell extract (e.g., invertebrate cells including, but not limited to, bacterial, fungal, algal, and plant cells) and synthetic sources, such as chemical compound libraries. For proteins with known activity, function assays are established based on the activity, and a large number of potential agents are screened for the ability to either stimulate or inhibit the activity. Binding assays are used to detect GPCR-like receptor binding activity to neuropeptide or non-peptide ligands. Both functional and binding assays of GPCR-like receptor activity are readily adapted to screens for modulators such as inhibitory compounds.
The invention contemplates a multitude of assays to screen and identify inhibitors of ligand binding to GPCR-like receptors. In one example, the GPCR-like receptor is immobilized and interaction with a binding partner is assessed in the presence and absence of a candidate modulator such as an inhibitor compound. In another example, interaction between the GPCR-like receptor and its binding partner is assessed in a solution assay, both in the presence and absence of a candidate inhibitor compound. In either assay, an inhibitor is identified as a compound that decreases binding between the GPCR-like receptor and its binding partner. Another contemplated assay involves a variation of the di-hybrid assay wherein an inhibitor of protein/protein interactions is identified by detection of a positive signal in a transformed or transfected host cell.
Candidate modulators contemplated by the invention include any chemical compounds, including libraries of chemical compounds. There are a number of different libraries used for the identification of small molecule modulators, including: (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules. Chemical libraries consist of random chemical structures, or analogs of known compounds, or analogs of compounds that have been identified as xe2x80x9chitsxe2x80x9d or xe2x80x9cleadsxe2x80x9d in prior drug discovery screens, some of which may be derived from natural products or from non-directed synthetic organic chemistry. Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see Science 282:63-68 (1998). Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture. These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or synthetic methods. Of particular interest are non-peptide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997). Identification of modulators through use of the various libraries described herein permits modification of the candidate xe2x80x9chitxe2x80x9d (or xe2x80x9cleadxe2x80x9d) to optimize the capacity of the xe2x80x9chitxe2x80x9d to modulate activity.
Candidate modulators contemplated by the invention can be designed and include soluble forms of binding partners, as well as chimeric, or fusion, proteins thereof. A xe2x80x9cbinding partnerxe2x80x9d as used herein broadly encompasses non-peptide modulators, peptide modulators (e.g., neuropeptide variants), antibodies (including monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, biftinctional/bispecific antibodies, humlianized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR and/or antigen-binding sequences, which specifically recognize a polypeptide of the invention), antibody fragments, and modified compounds comprising antibody domains that are immunospecific for the expression product of the identified GPCR-like gene.
A number of assays are known in the art that can identify chemical compounds that bind to or interact with a GPCR-like receptor. Such assays are useful, for example, in methods of identifying candidate modulators described herein, or in methods for identifying specific neuropeptide ligands of a GPCR-like receptor. Assays that measure binding or interaction of compounds with target proteins include assays that identify compounds that inhibit unfolding or denaturation of a target protein, assays that separate compounds that bind to target proteins through affinity ultrafiltration followed by ion spray mass spectroscopy/HPLC methods or other physical and analytical methods, capillary electrophoresis assays and two-hybrid assays.
One such screening method to identify direct binding of test ligands to a target protein is described in U.S. Pat. No. 5,585,277, incorporated herein by reference. This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states. When a test ligand binds to the folded form of a target protein (i.e., when the test ligand is a ligand of the target protein), the target protein molecule bound by the ligand remains in its folded state. Thus, the folded target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand. Binding of the ligand to the target protein can be determined by any method which distinguishes between the folded and unfolded states of the target protein. The function of the target protein need not be known in order for this assay to be performed. Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules.
Another method for identifying ligands of a target protein is described in Wieboldt et al., Anal. Chem., 69:1683-1691 (1997), incorporated herein by reference. This technique screens combinatorial libraries of 20-30 agents at a time in solution phase for binding, to the target protein. Agents that bind to the target protein are separated from other library components by simple membrane washing. The specifically selected molecules that are retained on the filter are subsequently liberated from the target protein and analyzed by HPLC and pneumatically assisted electrospray (ion spray) ionization mass spectroscopy. This procedure selects library components with the greatest affinity for the target protein, and is particularly useful for small molecule libraries.
Alternatively, such binding interactions are evaluated indirectly using the yeast two-hybrid system described in Fields et al., Nature, 340:245-246 (1989), and Fields et al., Trends in Genetics, 10:286-292 (1994), both of which are incorporated herein by reference. The two-hybrid system is a genetic assay for detecting interactions between two proteins or polypeptides. It can be used to identify proteins that bind to a known protein of interest, or to delineate domains or residues critical for an interaction. Variations on this methodology have been developed to clone genes that encode DNA binding proteins, to identify peptides that bind to a protein, and to screen for drugs. The two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast. The assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a first protein and (2) an activation domain fused to a second protein. The DNA-binding domain targets the first hybrid protein to the UAS of the reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription of the reporter gene. The second hybrid protein, which contains the activation domain, cannot by itself activate expression of the reporter gene because it does not bind the UAS. However, when both hybrid proteins are present, the noncovalent interaction of the first and second proteins tethers the activation domain to the UAS, activating transcription of the reporter gene.
When the function of the GPCR-like receptor is unknown and no ligands are known to bind the gene product, the yeast two-hybrid assay can be used to identify proteins that bind to the receptor. In an assay to identify proteins that bind to a GPCR-like receptor, or fragment thereof, a fusion polynucleotide encoding both a GPCR-like receptor or fragment (i.e., a first protein) and a UAS binding domain may be used. In addition, a large number of hybrid genes each encoding a different second protein fused to an activation domain are produced and screened in the assay. Typically, the second protein is encoded by one or more members of a total cDNA or genomic DNA fusion library, with each second protein coding region being fused to the activation domain. This system is applicable to a wide variety of proteins, and it is not even necessary to know the identity or function of the second binding protein. The system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mRNA that can be repeatedly translated to yield the reporter protein.
In addition, when the GPCR-like receptor or fragment thereof is known to interact with another protein or nucleic acid, the two-hybrid assay can be used to detect agents that interfere with the binding interaction. Expression of the reporter gene is monitored as different test agents are added to the system. The presence of an inhibitory agent, for example, results in lack of or reduction in a reporter signal.